Bacillus thuringiensis mutants which produce high yields of crystal delta-endotoxin

ABSTRACT

The invention relates to a mutant of Bacillus thuringiensis which produces a larger amount of crystal delta-endotoxin with a greater pesticidal activity as compared to the corresponding parental strain. The mutant may also have a larger crystal size as compared to the corresponding parental strain. The crystal delta-endotoxin produced by the mutant Bacillus thuringiensis will have an activity directed towards the same pest(s) as its parental Bacillus thuringiensis crystal delta-endotoxin. The invention further relates to a method for producing such a mutant, compositions comprising such a mutant as well as methods for controlling a pest(s) using these compositions.

This application is a continuation of U.S. patent application Ser. No.08/379,683, filed Jan. 25, 1995, now abandoned which is acontinuation-in-part application Ser. No. 08/182,904, filed Jan. 14,1994, now abandoned which is a continuation of application Ser. No.07/906,038, filed Jun. 26, 1992, now U.S. Pat. No. 5,279,962, which is acontinuation of application Ser. No. 07/613,337, filed Nov. 14, 1990,now abandoned.

1. FIELD OF THE INVENTION

The invention relates to a mutant of Bacillus thuringiensis whichproduces a larger amount of crystal delta-endotoxin with a greaterpesticidal activity as compared to the corresponding parental strain.The mutant may also have a larger crystal size as compared to thecorresponding parental strain. The crystal delta-endotoxin produced bythe mutant Bacillus thuringiensis will have an activity directed towardsthe same pest(s) as its parental Bacillus thuringiensis crystaldelta-endotoxin. The invention further relates to a method for producingsuch a mutant, compositions comprising such a mutant as well as methodsfor controlling a pest(s) using these compositions.

2. BACKGROUND OF THE INVENTION

Every year, pests detrimental to agriculture, forestry, and publichealth cause losses in the millions of dollars. Various strategies havebeen used in attempting to control such pests.

One strategy is the use of chemical pesticides with a broad range orspectrum of activity. However, there are a number of disadvantages tousing such chemical pesticides. Specifically, because of their broadspectrum of activity, these pesticides may destroy non-target organismssuch as beneficial insects and parasites of destructive pests.Additionally, chemical pesticides are frequently toxic to animals andhumans. Furthermore, targeted pests frequently develop resistance whenrepeatedly exposed to such substances.

Another strategy has involved the use of biopesticides, which make useof naturally occurring pathogens to control insect, fungal and weedinfestations of crops. An example of a biopesticide is a bacterium whichproduces a substance toxic to the infesting pest. A biopesticide isgenerally less harmful to non-target organisms and the environment as awhole than chemical pesticides.

The most widely used biopesticide is Bacillus thuringiensis. Bacillusthuringiensis is a motile, rod-shaped, gram-positive bacterium that iswidely distributed in nature, especially in soil and insect-richenvironments. During sporulation, Bacillus thuringiensis produces aparasporal crystal inclusion(s) which is insecticidal upon ingestion tosusceptible insect larvae of the order Lepidoptera, Diptera, orColeoptera. The inclusion(s) may vary in shape, number, and composition.They are comprised of one or more proteins called crystaldelta-endotoxins, which may range in size from 27-140 kDa. Theinsecticidal crystal delta-endotoxins are generally converted byproteases in the larval gut into smaller (truncated) toxic polypeptides,causing midgut destruction, and ultimately, death of the insect (Hofteand Whiteley, 1989, Microbiol. Rev. 53:242-255).

There are several Bacillus thuringiensis strains that are widely used asbiopesticides in the forestry, agricultural, and public health areas.Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp.aizawai have been found to produce crystal delta-endotoxins specific forLepidoptera. Bacillus thuringiensis subsp. israelensis has been found toproduce crystal delta-endotoxins specific for Diptera (Goldberg, 1979,U.S. Pat. No. 4,166,112). Bacillus thuringiensis subsp. tenebrionis(Krieg et al., 1988, U.S. Pat. No. 4,766,203), has been found to producea crystal delta-endotoxin specific for Coleoptera. Several Bacillusthuringiensis crystal delta-endotoxin proteins are also reportedlypesticidal to nematodes, Acari, Hymenoptera, Phthiraptera,Platyhelminthes, Homoptera, Blattodea, and Protozoa.

The isolation of another coleopteran toxic Bacillus thuringiensis strainwas reported in 1986 (Herrnstadt et al., 1986, Bio/Technology 4:305-308;Herrnstadt and Soares, 1988, U.S. Pat. No. 4,764,372). This strain,designated "Bacillus thuringiensis subsp. san diego", M-7, has beendeposited at the Northern Regional Research Laboratory, USA underaccession number NRRL B-15939. However, the assignee of the '372 patent,Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensissubsp. san diego is Bacillus thuringiensis subsp. tenebrionis.furthermore, the '372 patent has been assigned to Novo Nordisk A/S. Aspo⁻ cry⁺ (asporogenous crystal forming) mutant of M-7 has purportedlybeen obtained by culturing M-7 in the presence of ethidium bromide(Herrnstadt and Gaertner, 1987, EP Application No. 228,228). However,there was no indication of increased production of delta-endotoxin,increased parasporal crystal size, and/or increased pesticidal activityrelative to the parental, M-7 strain.

The delta-endotoxins are encoded by cry (crystal protein) genes. The crygenes have been divided into six classes and several subclasses based onrelative amino acid homology and pesticidal specificity. The six majorclasses are Lepidoptera-specific (cryI), Lepidoptera- andDiptera-specific (cryII), Coleoptera-specific (cryhIII),Diptera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiol. Rev.53:242-255), Coleoptera- and Lepidoptera-specific (referred to as cryVgenes by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); andNematode-specific (referred to as cryV and cryVI genes by Feitelson etal., 1992, Bio/Technology 10:271-275).

The utility of Bacillus thuringiensis strains for the control of pestsis dependent upon efficient and economical production of the activetoxins. This in turn is dependent upon the amount of crystaldelta-endotoxins which can be produced by fermentation of the activeBacillus thuringiensis strains.

Consequently a recognized need for products of improved strength exists.

One way to fulfill this need would be to concentrate the preparations.However, this would add considerably to the production cost incomparison to the savings obtained in storage and transportation.

A much more elegant solution would be to create mutants of existing B.t.strains which produce substantially larger amounts of delta-endotoxinand have a substantially higher amount of pesticidal activity comparedto its parental strain. Such mutants would give a more efficient andeconomical production of active delta-endotoxins and a possibility formanufacture of B.t. products with increased potency at equal or lowercost. This in turn would be an advantage for the user as reduced volumesof pesticide formulation have to be stored and handled for a givenacreage. In addition, the users will have less container material todispose of, thereby reducing the impact on the environment.

3. SUMMARY OF THE INVENTION

The invention is directed to a mutant that produces larger amounts ofcrystal delta-endotoxin than the corresponding parental strain,preferably more than about 1.25 times and most preferably more thanabout 1.5 times the amount with greater pesticidal activity and hasactivity directed towards the same pest as a corresponding parentalstrain.

As defined herein, a "parental strain" is the original Bacillusthuringiensis strain before mutagenesis. In a specific embodiment, theparental strain is a wild-type strain.

"Greater Pesticidal activity" as defined herein means at least 1.25times and preferably more than about 1.5 more activity against a pest,times through killing or stunting of the growth of the pest, than thecorresponding parental strain. In a preferred embodiment, the pesticidalactivity of the mutant is between about 1.5 to about 5 times greaterthan the pesticidal activity of the corresponding parental Bacillusthuringiensis strain.

In a specific embodiment, the mutant may also have a larger crystalvolume, preferably more than 1.25 times and most preferably more thantwice the volume than the corresponding parental strain. The volume maybe determined by photographing spore/crystal preparations using amicroscope with a camera attachment. Measurements of the crystals inmillimeters may be made, and then normalized to the average length ofthe spores in each photo to account for any differences in photoenlargement. The volume for the bipyramidal crystals is calculated usingthe following formula: V=(width² ×length)/3. Additionally, the mutantsmay have a sporulation frequency of at least 2 logs lower than thesporulation frequency of the parental strain.

The invention further relates to a method for obtaining the mutants ofthe present invention. The invention also relates to a pesticidalcomposition comprising such a mutant or spore thereof and a pesticidallyacceptable carrier as well as methods for controlling a pest using sucha composition.

4. DETAILED DESCRIPTION OF THE INVENTION

The mutants of the present invention may have activity against an insectpest (e.g., lepidopteran, coleopteran, dipteran), as well as snails,mites, or nematodes. The parental Bacillus thuringiensis may be awild-type strain which includes but is not limited to Bacillusthuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. aizawai,Bacillus thuringiensis subsp. galleriae, Bacillus thuringiensis subsp.entomocidus, Bacillus thuringiensis subsp. tenebrionis, Bacillusthuringiensis subsp. thuringiensis, Bacillus thuringiensis subsp.alesti, Bacillus thuringiensis subsp. canadiensis, Bacillusthuringiensis subsp. darmstadiensis, Bacillus thuringiensis subsp.dendrolimus, Bacillus thuringiensis subsp. finitiimus, Bacillusthuringiensis subsp. kenyae, Bacillus thuringiensis subsp. morrisoni,Bacillus thuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp.toumanoffi, Bacillus thuringiensis subsp. toumanoffi, Bacillusthuringiensis subsp. pondicheriensis, Bacillus thuringiensis subsp.shandogiensis, Bacillus thuringiensis subsp. sotto, Bacillusthuringiensis subsp. nigeriae, Bacillus thuringiensis subsp.yunnanensis, Bacillus thuringiensis subsp. dakota, Bacillusthuringiensis subsp. nidiana, Bacillus thuringiensis subsp. tohokuensis,Bacillus thuringiensis subsp. kumamotoensis, Bacillus thuringiensissubsp. tochigiensis, Bacillus thuringiensis subsp. thompsoni, Bacillusthuringiensis subsp. wuhanensis, Bacillus thuringiensis subsp.kyushuensis, Bacillus thuringiensis subsp. ostriniae, Bacillusthuringiensis subsp. tolworthi, Bacillus thuringiensis subsp. pakistani,Bacillus thuringiensis subsp. japonensis, Bacillus thuringiensis subsp.colmeri, Bacillus thuringiensis subsp. pondicheriensis, Bacillusthuringiensis subsp. shandongiensis, Bacillus thuringiensis subsp.neoleonensis, Bacillus thuringiensis subsp. coreanensis, Bacillusthuringiensis subsp. silo, Bacillus thuringiensis subsp. mexcanensis,and Bacillus thuringiensis subsp. israelensis.

In a specific embodiment, the parental Bacillus thuringiensis strain isBacillus thuringiensis subsp. aizawai. In a most specific embodiment,the mutant has the identifying characteristics of EMCC0125, depositedwith the NRRL and having the accession number NRRL B-21389.

In another embodiment, the parental strain may solely produce aCryIA(a)-like crystal delta-endotoxin. Such strains are disclosed inapplication Ser. No. 08/157,363, filed Nov. 23, 1993, incorporatedherein by reference. The CryIA(a)-like crystal delta-endotoxin isencoded by at least one copy of a cryIA(a)-like gene. As defined herein,a "cryIA(a)-like genes is a DNA sequence encoding a CryIA(a)-likeprotein defined above. In a specific embodiment, the cryIA(a)-like genehas at least 90% homology to the cryIA(a) gene, preferably at least 95%homology to the cryIA(a) gene and most preferably at least 99% homologyto the cryIA(a) gene and is shown in the sequence listing as SEQ ID NO:1shown below.

CCTGGGTCAAAAATTGATATTTAGTAAAATTAGTTGCACTTTGTGCATTTTTTCATAAGATGAGTCATATGTTTTAAATTGTAGTAATGAAAAACAGTATTATATCATAATGAATTGGTATCTTAATAAAAGAGATGGAGGTAACTTATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTGGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGATTATGCTGTGCGCTGGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGATTGGGTAAGGTATAATCAATTTAGAAGAGAGCTAACACTTACTGTATTAGATATCGTTGCTCTATTCTCAAATTATGATAGTCGAAGGTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACGAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGTGGAATGGCTCAGAGAATAGAACAGAATATTAGGCAACCACATCTTATGGATATCCTTAATAGTATAACCATTTATACTGATGTGCATAGAGGCTTTAATTATTGGTCAGGGCATCAAATAACAGCTTCTCCTGTAGGGTTTTCAGGACCAGAATTCGCATTCCCTTTATTTGGGAATGCGGGGAATGCAGCTCCACCCGTACTTGTCTCATTAACTGGTTTGGGGATTTTTAGAACATTATCTTCACCTTTATATAGAAGAATTATACTTGGTTCAGGCCCAAATAATCAGGAACTGTTTGTCCTTGATGGAACGGAGTTTTCTTTTGCCTCCCTAACGACCAACTTGCCTTCCACTATATATAGACAAAGGGGTACAGTCGATTCACTAGATGTAATACCGCCACAGGATAATAGTGTACCACCTCGTGCGGGATTTAGCCATCGATTGAGTCATGTTACAATGCTGAGCCAAGCAGCTGGAGCAGTTTACACCTTGAGAGCTCCAACGTTTTCTTGGCAGCATCGCAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAGAATTCGCTACGCTTCTACTACAAATTTACAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGTAATTTTTCAGCAACTATGAGTAGTGGGAGTAATTTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTTCCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACAGATGTGACGGATTATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCAGATGAATTTTGTCTGGATGAAAAACAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAACTTCAGAGGGATCAATAGACAACTAGACCGTGGCTGGAGAGGAAGTACGGATATTACCATCCAAGGAGGCGATGACGTATTCAAAGAGAATTACGTTACGCTATTGGGTACCTTTGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTATCAATTAAGAGGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTACAATGCAAAACATGAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGGCCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAGCCGAATCGATGCGCGCCACACCTTGAATGGAATCCTGACTTAGATTGTTCGTGTAGGGATGGAGAAAAGTGTGCCCATCATTCGCATCATTTCTCCTTAGACATTGATGTAGGATGTACAGACTTAAATGAGGACCTAGGTGTATGGGTGATCTTTAAGATTAAGACGCAAGATGGGCACGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTAGTAGGAGAAGCGCTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGGGAAACAAATATCGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTTGTAAACTCTCAATATGATCAATTACAAGCGGATACGAATATTGCCATGATTCATGCGGCAGATAAACGTGTTCATAGCATTCGAGAAGCTTATCTGCCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTGAAAGGCATGTAGATGTAGAAGAACAAAACAACCAACGTTCGGTCCTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTTAGCAACTGCGTAGAAGAGGAAATCTATCCAAATAACACGGTAACGTGTAATGATTATACTGTAAATCAAGAAGAATACGGAGGTGCGTACACTTCTCGTAATCGAGGATATAACGAAGCTCCTTCCGTACCAGCTGATTATGCGTCAGTCTATGAAGAAAAATCGTATACAGATGGACGAAGAGAGAATCCTTGTGAATTTAACAGAGGGTATAGGGATTACACGCCACTACCAGTTGGTTATGTGACAAAAGAATTAGAATACTTCCCAGAAACCGATAAGGTATGGATTGAGATTGGAGAAACGGAAGGAACATTTATCGTGGACAGCGTGGAATTACTCCTTATGGAGGAATAGTCTCATGCAAACTCAGGTTTAAATATCGTTTTCAAATCAATTGTCCAAGAGCAGCATTACAAATAGATAAGTAATTTGTTGTAATGAAAAACGGACATCACCTCCATTGAAACGGAGTGATGTCCGTTTTACTATGTTATTTTCTAGT

In a specific embodiment, the parental strain may be the B.t. strainEMCC0073 or EMCC0074 also disclosed in application Ser. No. 08/157,363.In a most specific embodiment, the mutant may have the identifyingcharacteristics of EMCC0123, deposited with the NRRL and having theaccession number NRRL B-21387 and the mutant or the identifyingcharacteristics of EMCC0124 and having the accession number NRRLB-21388.

4.1. METHODS OF OBTAINING THE MUTANT

The parental strain may be treated with a mutagen to induce atransposition event. Due to the nature of transposition, suchmutagenesis will also cause a gene duplication, in this case,duplication of the cry gene. Specifically, in one method of mutatingBacillus thuringiensis strains and selecting such mutants that arecapable of producing substantially larger amounts of crystaldelta-endotoxins than their parental strains, the parental strain is:

i) treated with a mutagen,

ii) the treated cells are cultured in a selective culture medium (e.g.,NSMP medium); and

iii) cells are selected which have a larger crystal size.

In step (i), the mutagen for example may be a chemical mutagenN-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate, gammairradiation, X-ray or UV-irradiation.

The cells are selected visually by examining the cultured cells under amicroscope. Cells which appear to have a larger crystal size are thusselected and delta-endotoxin production and activity is assayed.

In one embodiment, the following procedure is used after treating cellswith mutagen:

i) growing mutagenized cells on a medium suitable for the selection ofasporogenous and/or oligosporogenous strains,

ii) selecting translucent colonies and growing the selected cells in amedium that does not fluidize on heating, and

iii) deselecting truly asporogenous strains by subjecting the coloniesto a heat treatment.

After deselection, the thus selected colonies are grown in a normalproduction medium, and a final selection for strains capable ofincreasing the crystal delta-endotoxin production is performed.

In step (i), a suitable medium could be a modified nutrient sporulationmedium including phosphate (NSMP medium) as described by Johnson et al.,In "Spores VI": eds. P. Gerhardt et al., pp. 248-254, 1975.

In step (iii) of the method of the invention, a suitable medium could bea NSMP medium supplemented with MgCl₂ and Gelrite.

Another method of obtaining the high producing mutants of the inventionmay be contemplated such as growing the parent strain in a liquid mediumand selecting spontaneous mutants after spreading the culture broth onan agar medium suitable for selection of asporogenous and/oroligosporogenous mutants.

Other methods of screening for the high producing mutants of theinvention may be contemplated such as separating the mutants from othermaterial on the basis of mass directly through centrifugation or othermeans of separating for mass.

4.2. COMPOSITIONS

The mutant Bacillus thuringiensis strains, crystal delta-endotoxinsand/or spores of the invention, can be formulated into a pesticidalcomposition(s), that is for example, a suspension, a dispersion, anaqueous emulsion, a dusting powder, a dispersible powder, anemulsifiable concentrate, an aerosol or micro or macroencapulatedgranules or any other formulation that gives controlled release ofBacillus thuringiensis. Such compositions may be obtained by theaddition of a surface active agent, e.g., a dispersing agent,emulsifying agent or wetting agent, or an inert carrier or othercomponent to facilitate handling and application for particular targetpests.

Suitable surface-active agents include anionic compounds such as acarboxylate, for example, a metal carboxylate of a long chain fattyacid; a N-acylsarcosinate; mono or di-esters of phosphoric acid withfatty alcohol ethoxylates or salts of such esters; fatty alcoholsulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate orsodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylatedalkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkylaryl sulphonates such as alkyl-benzene sulphonates or loweralkylnaphthalene sulphonates, e.g., butyl-naphthalene sulphonate; saltsor sulphonated naphthalene-formaldehyde condensates or salts ofpolyacrylic acid; salts of sulphonated phenol-formaldehyde condensates;or more complex sulphonates such as the amide sulphonates, e.g., thesulphonated condensation product of oleic acid and N-methyl taurine orthe dialkyl sulphosuccinates, e.g., the sodium sulphonate or dioctylsuccinate. Non-ionic agents include condensation products of fatty acidesters, fatty alcohols, fatty acid amides or fatty-alkyl- oralkenyl-substituted phenols with ethylene oxide and/or propylene oxide,fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acidesters, condensation products of such esters with ethylene oxide, e.g.,polyoxyethylene sorbitar fatty acid esters, block copolymers of ethyleneoxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate;an oxygen-containing amine such as an amine oxide of polyoxyethylenealkylamine; an amide-linked amine prepared by the condensation of acarboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

Examples of inert materials include inorganic minerals such asphyllosilicates, carbonates, sulfates, phosphates; organic materialssuch as sugar, starches, or cyclodextrins; or botanical materials suchas powdered corncobs, rice hulls, walnut shells, cornmeal, pelletedgrains, and cellulosic fibers.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate or primary composition whichrequires dilution with a suitable quantity of water or other diluentbefore application. The pesticidal concentration will vary dependingupon the nature of the particular formulation, specifically, whether itis a concentrate or to be used directly. The composition contains 0.1%to 99%, preferably 0.1% to 95% of the mutant, mutant or variant of thepresent invention, 1 to 98% of a solid or liquid inert carrier, and 0 to50%, preferably 0.1% to 50% of a surfactant. These compositions will beadministered at about 0.01 lb-5.0 lb per acre when in dry form and atabout 0.01 pt-10 pts per acre when in liquid form.

In a further embodiment, the mutants of the present invention can betreated prior to formulation to prolong the pesticidal activity when thecells are applied to the environment of a target pest. Such treatmentcan be by chemical and/or physical means as long as the treatment doesnot deleteriously affect the properties of the composition(s). Examplesof chemical reagents include, but are not limited to, halogenatingagents; aldehydes such as formaldehyde and glutaraldehyde;anti-infectives, such as zephiran chloride; alcohols, such asisopropranol and ethanol; histological fixatives, such as Bouinmsfixative and Helly's fixative (see, for example, Humason, Animal TissueTechniques, W. H. Freeman and Co., 1967); preservatives; UV sunscreens;spray adjuvants (humectants); antifoams; and stickers.

The compositions of the invention can be applied directly to the plantby, for example, spraying or dusting at the time when the pest has begunto appear on the plant or before the appearance of pests as a protectivemeasure. Plants to be protected within the scope of the presentinvention include, but are not limited to, cereals (wheat, barley, rye,oats, rice, sorghum and related crops), beet (sugar beet and fodderbeet), drupes, pomes and soft fruit (apples, pears, plums, peaches,almonds, cherries, strawberries, raspberries, and blackberries,tomatoes), leguminous plants (beans, lentils, peas, soybeans), oilplants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oilplants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows,melons), fibre plants (cotton, flax, hemp, jute), citrus fruit (oranges,lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus,cabbages and other brassicae, carrots, onions, potatoes, paprika),lauraceae (avocados, cinnamon, camphor), deciduous trees and conifers(linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs,larches, pines), or plants such as maize, tobacco, nuts, coffee, sugarcane, tea, vines hops, bananas and natural rubber plants, as well asornamentals. The preferred mode of application is by foliar spraying. Itis generally important to obtain good control of pests in the earlystages of plant growth as this is the time when the plant can be mostseverely damaged. The spray or dust can conveniently contain anotherinsecticide or pesticide, e.g., fungicide, grass herbicide orfertilizer, if this is thought necessary. In a preferred embodiment, thecomposition of the invention is applied directly to the plant.

The compositions of the present invention may be effective against pestsof the order Lepidoptera, e.g., Achroia grisella, Acleris gloverana,Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea,Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsialineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,Archrps sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrixthurberiella, Cadra cautella, Choristoneura sp., Cochylis hospes, Coliaseurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella,Datana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphaniahyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella,Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupocoelliaambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileucaoliviae, Homoeosoma electellum, Hyphantria cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis,Lymantria dispar, Macalla thyrsisalis, Malacosoma sp., Mamestrabrassicae, Mamestra configurata, Manduca cuinquemaculata, Manduca sexta,Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota sultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp.,Syngrapha falcifera, Thaurnstopoea pityocampa, Tineola bisselliella,Trichoplusia ni, Udea rubigalis, xylomyges curialis, Yponomeutapadella;. The compositions of the invention may also be effectiveagainst insect pests of the order Coleoptera, e.g., Leptinotarsa sp.,Acanthoscelides obtectus, Callosobruchus chinensis, Epilachnavarivestis, Pyrrhalta luteola, Cylas formicarius elegantulus,Listronotus oregonensis, Sitophilus sp., Cyclocephala borealis,Cyclocephala immaculata, Macrodactylus subspinosus, Popillia japonica,Rhizotrogus majalis, Alphitobius diaperinus, Palorus ratzeburgi,Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum, Triboliumconfusum, Tribolius destructor, Diptera, e.g., Aedes sp., Andesvittatus, Anastrepha ludens, Anastrepha suspensa, Anopheles barberi,Anopheles quadrimaculatu's, Armigeres subalbatus, Calliphora stygian,Calliphora vicina, Ceratitis capitata, Chironomus tentans, Chrysomyarufifacies, Cochliomyia macellaria, Culex sp., Culiseta inornata, Dacusoleae, Delia antiqua, Delia platura, Delia radicum, Drosophilamelanogaster, Eupeodes corollae, Glossina austeni, Glossina brevipalpis,Glossina fuscipes, Glossina morsitans centralis, Glossina morsitansmorsitans, Glossina morsitans submorsitans, Glossina pallidipes,Glossina palpalis gambiensis, Glossina palpalis palpalis, Glossinatachinoides, Haemagogus equinus, Haematobia irritans, Hypoderma bovis,Hypoderma lineatum, Leucopis ninae, Lucilia cuprina, Lucilia sericata,Lutzomyia longlpaipis, Lutzomyia shannoni, Lycoriella mali, Mayetioladestructor, Musca autumnalis, Musca domestica, Neobellieria sp.,Nephrotoma suturalis, Ophyra aenescens, PhaenicIa sericata, Phlebotomussp., Phormia regina, Sabethes cyaneus, Sarcophaga bullata, Scatophagastercorarla, Stomaxys calcitrans, Toxorhynchites amboinensis,Tripteroides bambusa; Acari, e.g., Oligonychus pratensis, Panonychusulmi, Tetranychus urticae; Hymenoptera, e.g., Iridomyrmex humilis,Solenopsis invicta; Isoptera, e.g., Reticulitermes hesperus,Reticulitermes flavipes, Coptotermes formosanus, Zootermopsisangusticollis, Neotermes connexus, Incisitermes minor, Incisitermesimmigrans; Siphonaptera, e.g., Ceratophyllus gallinae, Ceratophyllusniger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalides canis,Ctenocephalides felis, Echicnophaga gallinacea, Pulex irritans,Xenopsylla cheopis, Xenopsylla vexabilis, Tunga penetrans; andTylenchida, e.g., Melodidogyne incognita, Pratylenchus penetrans.

The following examples are presented by way of illustration, not by wayof limitation.

EXAMPLES

5.1. Example 1

A mutant of B. thuringiensis subsp. tenebrionis with more than a twofoldincrease in crystal delta-endotoxin production has been isolated. Phasecontrast microscopy, scanning electron microscopy and transmissionelectron microscopy of this mutant indicate that the high productivityof this mutant is due to changes in the regulation of crystaldelta-endotoxin production relative to sporulation resulting in theproduction of protein crystals which are up to more than five timesbigger than the crystals produced by the known coleopteran activeBacillus thuringiensis strains. The close correlation between crystalformation and sporulation seems to have been removed and the mutantproduces high amounts of crystal delta-endotoxin prior to sporulation.

5.1.1. Production of High Yield Mutant

Spores of B. thuringiensis subsp. tenebrionis, strain DSM 5526 aregamma-irradiated to give a dosage of 7 kGy. The irradiated spores arespread onto NSMP agar plates (modified nutrient sporulation mediumincluding phosphate as described by Johnson et al., In "Spores VI": eds.P. Gerhardt et al., pp. 248-254, 1975), a medium suitable for selectionof asporogenous and/or oligosporogenous mutants.

The NSMP-agar plates are incubated at 30° C. for 2-3 days. Translucentcolonies are picked out and transferred to NSMP gelrite plates (NSMPmedium supplemented with MgCl₂ (0.57 g/l) and Gelrite, Kelco (20 g/l)).

The NSMP gelrite plates are incubated for one hour at 90° C. and thenfurther incubated for 1-2 days at 30° C. Mutants that grew well on theNSMP gelrite plates are selected. In this way, all asporogenous mutantsare deselected as they fail to grow after the heat treatment.

The selected mutants are grown in shakeflasks containing a commercialmedium. The amounts of crystal delta-endotoxin produced are determinedby immunological methods described below. Only mutants producingsignificantly higher amounts of crystal delta-endotoxin than the parentstrain are selected.

The morphology of the selected mutants on solid medium and in liquidmedia are studied by phase contrast microscopy (×2500) and by scanningand transmission eledtron microscopy. The number of spores and crystalsare counted and the size of the protein crystals are determined. Amongthe mutants obtained, one (DSM 5480) is selected for its outstandingability to produce crystal delta-endotoxin.

The amount of crystal delta-endotoxin produced by mutant DSM 5480 iscompared with that of DSM 2803 the original isolate of Bacillusthuringiensis subsp. tenebrionis, Bacillus thuringiensis subsp.tenebrionis, strain DSM 5526, Bacillus thuringiensis subsp. tenebrionis,strain NB178, isolated from Sandoz' Bacillus thuringiensis tenebrionisproduct TRIDENT® from 1989, strain NB 198, isolated from Sandoz' B.thuringiensis subsp. tenebrionis product TRIDENT® from 1990, "Bacillusthuringiensis subsp. san diego", strain NRRL-B-15939, and strain NB 197isolated from Mycogen's B. thuringiensis subsp. san diego" productM-ONE® from 1990. As shown in Table I of Example 2, the yield improvedmutant of the invention produces 2-3.5 times as much crystaldelta-endotoxin as the coleopteran active strains of Bacillusthuringiensis available today.

5.2. Example 2

In this example, the crystal delta-endotoxin yield of Bacillusthuringiensis subspecies tenebrionis, mutant DSM 5480 is compared withthe crystal delta-endotoxin yields of Bacillus thuringiensis subspeciestenebrionis strains DSM 2803 (the original isolate of Bacillusthuringiensis subsp. tenebrionis), DSM 5526 (a production strain ofNovo-Nordisk) and NB 178 and NB 198 (production strains of Sandoz), andBacillus thuringiensis subsp. san diego, strain NRRL-B 15939 and NB 197(production strains of Mycogen) in a commercial medium. Each of thestrains is grown for 17 hours at 30° C. on agar slants of the followingcomposition expressed as gram per liter of distilled water.

    ______________________________________    Peptone, Difco         5     g    Beef extract, Difco    3     g    Agar, Difco            20    g    pH                     7.0    ______________________________________

5 ml of a suspension of cells from each strain are then transferred to100 ml of production medium in 500 ml baffle-bottom Erlenmeyer flasks.The production medium consisted of the following components in thequantities indicated (expressed as grams per liter of tap water).

    ______________________________________    Soy bean meal         50     g    Hydrolyzed starch     40     g    KH.sub.2 PO.sub.4     1.77   g    K.sub.2 HPO.sub.4     4.53   g    pH                    7.0    ______________________________________

The inoculated flasks are incubated at 30° C. with shaking (250 rpm).After 96 hours of incubation the culture broths are assayed for crystaldelta-endotoxin yields by immunological methods.

The amounts of crystal delta-endotoxin produced by the individualstrains are determined by rocket immunoelectrophoresis (RIE) and aphotometric immunoassay (PIA) using antibodies raised against purifiedprotein crystals from Bacillus thuringiensis subsp. tenebrionis.

400 mg of each culture broth are weighed. 7 ml trisodium phosphatebuffer (0.125M, pH 12) is added to each sample. The suspensions areshaken for 1 hour in order to solubilize the crystal delta-endotoxinproteins.

The samples are then centrifuged at 3,500 rpm for 15 minutes and thesupernatants are tested for crystal delta-endotoxin by rocketimmunoelectrophoresis against antiserum raised against purified proteincrystals from B. thuringiensis subsp. tenebrionis. The amounts ofcrystal delta-endotoxin are determined relatively to a standard withknown content of crystal protein.

The concentration of crystal delta-endotoxin is also determined by aphotometric immunoassay. The crystal delta-endotoxins are dissolved inan alkaline solution. The dissolved proteins are precipitated by theirantibodies. The rate of this reaction is determined turbidimetrically.The amounts of crystal delta-endotoxin are determined relatively to astandard with known content of crystal protein.

Crystal delta-endotoxins for production of the antibodies used in theassays are obtained from crystals isolated from B. thuringiensis subsp.tenebrionis. Polyclonal antibodies are raised by injecting rabbitssubcutaneously every fortnight with 0.25 mg of crystal delta-endotoxin.

The results obtained are shown in the following Tables Ia and Ib.Crystal delta-endotoxin yields are expressed as BTTU/g (units per gculture broth, determined by rocket immunoelectrophoresis, RIE, or by aphotometric immunoassay, PIA). The value used for pure B. thuringiensissubsp. tenebrionis crystal delta-endotoxin is 500,000 BTTU/g. The valuesindicated in Table Ia below being averages of 6-7 independentfermentations, and those in Table Ib being averages of 3 independentfermentations.

                  TABLE Ia    ______________________________________    Crystal delta-endotoxin production by strains of Bacillus    thuringiensis subsp. tenebrionis in shake flasks                   Crystal delta-endotoxin                   yield                     RIE     PIA    Strain           BTTU/g  BTTU/g    ______________________________________    DSM 2803         676     1293    NRRL-B 15939     747     1126    NB 178           986     1728    DSM 5526         1097    1860    DSM 5480         2382    4169    ______________________________________

                  TABLE Ib    ______________________________________    Crystal delta-endotoxin production by strains of Bacillus    thuringiensis subsp. tenebrionis in shake flasks                Crystal delta-endotoxin yield                RIE    Strain      BTTU/g    ______________________________________    NB 197      1103    NB 198      1237    DSM 5480    2867    ______________________________________

From Tables Ia and Ib, it appears that DSM 5480 produces more than threetimes as much crystal delta-endotoxin as the original strain of Bacillusthuringiensis subsp. tenebrionis, DSM 2803 and "Bacillus thuringiensissubsp. san diego", strain NRRL-B15939 and more than twice the amount ofcrystal delta-endotoxin as the strains used today for the manufacture ofcommercial products of Bacillus thuringiensis subsp. tenebrionis.

Phase contrast microscopy, scanning electron microscopy and transmissionelectron microscopy of Bacillus thuringiensis subsp. tenebrionis, mutantDSM 5480 have revealed that the protein crystals produced by this mutantare much bigger than the corresponding protein crystals produced byBacillus thuringiensis subsp. tenebrionis, strains DSM 2803, DSM 5526,NB178 and NB 198, and "Bacillus thuringiensis subsp. san diego", strainsNRRL-B 15939 and NB 197.

Culture broth of Bacillus thuringiensis subsp. tenebrionis, mutant DSM5480 is tested for activity against Leptinotarsa texana larvae. Theincreased amount of crystal delta-endotoxin produced by mutant DSM 5480as determined by the immunological methods is reflected in thebiological activity against Leptinotarsa texana larvae.

5.3. Example 3

In this example, sporulation and parasporal crystal formation in B.thuringiensis subsp. tenebrionis, strains, DSM 2803, DSM 5526, NB 178and NB 198, and mutant DSM 5480, and "B. thuringiensis subsp. sandiego", strains NRRL-B 15939 and NB 197 are compared on solid medium andin liquid medium.

Each of the strains is grown for 2 days at 30° C. on agar plates of thefollowing composition expressed as gram per liter of distilled water.

    ______________________________________    Peptone, Difco         5     g    Beef extract, Difco    3     g    Agar, Difco            20    g    pH                     7.0    ______________________________________

Each of the strains is also grown in liquid medium. All strains aregrown for 17 hours at 30° C. on agar slants. 5 ml of a suspension ofcells from each strain are then transferred to 500 ml baffle bottomErlenmeyer flasks each containing 100 ml of medium.

The medium consisted of the following components in the quantitiesindicated (expressed as grams per liter of tapwater).

Liquid medium:

    ______________________________________           Yeast extract  5     g           Tryptone       5     g           Glucose        1     g           KH.sub.2 PO.sub.4                          0.8   g           pH             7.0    ______________________________________

The inoculated flasks are incubated at 30° C. with shaking (250 rpm) for96 hours.

The morphology of the strains on the solid medium and in the liquidmedium is studied daily by phase contrast microscopy (×2500). The numberof spores and crystals are counted and the size of the parasporalcrystals are determined. A few selected samples are also studied byscanning and transmission electron microscopy.

B. thuringiensis subsp. tenebrionis, strains DSM 2803, DSM 5526, NB 178and NB 198, and "B. thuringiensis subsp. san diego", strains NRRL-B15939 and NB 197 all sporulated well on both media. Before cell lysis,each cell contained a spore and a parasporal crystal. The size of thecrystals is from 0.4 to 0.9-1.1 μm in length by the time of cell lysis,the average size of the protein crystals being 0.6-0.7 μm in length.

Mutant DSM 5480 produced only few spores (<10⁶ spores/ml) on the solidmedium and in the defined liquid medium. Before cell lysis most cellscontained a huge protein crystal but no spore. The size of the proteincrystals is from 0.4-0.7 μm to 5.0 μm, the average size of the proteincrystals being 2.2-2.3 μm in length.

Ultrastructural analysis of cells from these media by transmissionelectron microscopy revealed that the sporulation process in the mutanthad been started but had not been completed by the time of cell lysis.The sporulation process had reached different stages in the variouscells. In cells where the sporulation had only reached stage II(forespore septum formation), the protein crystals filled up the entirecells.

In the production medium (Example 2) the mutant produced a higher numberof spores (10⁷ -10⁸ spores/ml). In this medium, the sporulationfrequency of the mutant is 10-100 times lower than in the parent strain.

Thus, the mutant has retained its ability to produce normal spores.However, the sporulation frequency of the mutant seems to be stronglydependent on the media.

The size of the protein crystals produced by the individual strains areshown in Tables IIa and IIb.

                  TABLE IIa    ______________________________________    Size of the protein crystals produced by coleopteran    active B. thuringiensis strains available today.              Length of protein crystals in μm                Minimum      Maximum  Mean    Strain      value        value    value    ______________________________________    DSM 2803    0.4          0.9      0.7    NRRL-B-15939                0.4          0.9      0.7    NB 178      0.5          0.9      0.7    DSM 5526    0.4          0.9      0.7    DSM 5480    0.7          5.0      2.3    ______________________________________

                  TABLE IIb    ______________________________________    Size of the protein crystals produced by coleopteran    active B. thuringiensis strains available today.            Length of protein crystals in μm              Minimum       Maximum  Mean    Strain    value         value    value    ______________________________________    NB 197    0.4           0.7      0.6    NB 198    0.4           1.1      0.7    DSM 5480  0.4           4.2      2.0    ______________________________________

From Tables IIa and IIb it is clear that mutant DSM 5480 produces muchbigger protein crystals than other coleopteran active B.t. strains.

From the data obtained, it appears that the regulation of crystaldelta-endotoxin production in relation to sporulation has been changedin the mutant. The mutant seems to produce the crystal delta-endotoxinprior to the development of spores hereby giving the cells a longerperiod for crystal delta-endotoxin production which result in theproduction of much bigger protein crystals by the time of cell lysisthan in the parent strain. Depending on the available nutrients and thesize of the crystal delta-endotoxins in the cells by the time ofsporulation, a normal spore will be developed before the time of celllysis.

5.4. Example 4

In this example, the high yielding BEtt mutant DSM 5480 is used toproduce high potency products for the control of Leptinotarsa texanalarvae.

DSM 5480 is fermented on the production fermentation medium described inExample 2 in an aerated, stirred production fermentation tank. After 96hours, the broth is recovered by centrifugation on a continuouscentrifuge. The concentrated cream which contains the active proteincrystals is stabilized by addition of microbial preservatives and pH isadjusted to 5.0. One portion of the concentrated cream is spray driedand later used for the formulation of wettable powder. The rest of theconcentrated cream is used directly for formulation of two aqueousflowable concentrates (FC). The wettable powder is formulated asdescribed in Table III. The formulation of the two FC's is described inTable IV.

                  TABLE III    ______________________________________    NOVODOR ® wettable powder formulation    Component            % by weight    ______________________________________    Spray dried concentrated cream of Btt                         40    Detergents           9    Anticaking agent     1    Inert filler         50    ______________________________________

                  TABLE IV    ______________________________________    NOVODOR ® FC formulations                 NOVODOR ® FC 1                               NOVODOR ® FC 2    Component    % by weight   % by weight    ______________________________________    Btt concentrated cream                 80            55    Preservatives                 4             4    Antifreeze agents                 9.1           19    Detergents   2.5           2.5    pH regulator 2.85          2.85    water        1.55          16.65                 100.0         100.0    ______________________________________

When using a value of 500,000 BTTU/g of pure crystal protein the contentof active crystal protein in the formulations are:

    ______________________________________                       % Btt crystal protein    ______________________________________    NOVODOR ® WP                  70.8 KBTTU/g                             14.16    NOVODOR ® FC1                  24.7 KBTTU/g                             4.94    NOVODOR ® FC2                  14.2 KBTTU/g                             2.84    ______________________________________

The detergents are chosen among the wide selection of suspension aidsand wetting agents normally used in agricultural pesticide products. Theanticaking agent is a hydrophilic silica and the inert filler is chosenfrom the generally used inert fillers such as bentonites, inorganicsalts or clays. The preservatives used in the FC's are chosen from thegroup of food and cosmetic preservatives. The pH regulator is aninorganic acid.

5.5. Example 5

A field trial is conducted to prove the biological effect of the highyielding Btt mutant DSM 5480 on the main target pest, Leptinotarsatexana larvae. Yields are compared with those obtained from the twocommercial products, Trident® and M-one®. The crop is potatoes.

The crop is sprayed 3 times on Jul. 20th, Jul. 27th and Aug. 3rd (2ndgeneration larvae). The products and dosages used are:

    ______________________________________                                   % Btt crystal               Product   Potency   protein in the               volume/acre                         KBTTU/g   formulation    ______________________________________    NOVODOR ® FC 2                 1      qt/acre  14.2    2.84                 1.5    qts/acre 14.2    2.84                 2.5    qts/acre 14.2    2.84                 3.0    qts/acre 14.2    2.84    TRIDENT ®                 4      qts/acre 5.5     1.10    M-one ®  2      qts/acre 8.9     1.78    ______________________________________

The mean % control of CPB larvae compared to the untreated control isgiven in Table V. The Leptinotarsa texana pressure is very heavy in theuntreated control: 370 larvae per 20 plants on Aug. 1st and 904 larvaeper 20 plants on Aug. 8th.

                  TABLE V    ______________________________________                      % control    Treatment           August 1st                                 August 8th    ______________________________________    NOVODOR ® FC 2                   1     qt     99     99    NOVODOR ® FC 2                   1.5   qts    95     100    NOVODOR ® FC 2                   2.5   qts    98     99    NOVODOR ® FC 2                   3     qts    100    100    TRIDENT ®  4     qts    94     98    M-one ®    2     qts    98     98    ______________________________________

These results clearly show that products made with the high yieldingmutant DSM 5480 are effective for the control of colorado potato beetlelarvae in the field. The crystal delta-endotoxin produced by the highyielding strain is fully active as 1.5 qts NOVODOR® FC give as goodresults as Trident at 4 qts and as good as M-one at 2 qts.

5.6. Example 6

Classical mutagenesis of Bacillus thuringiensis CryIA(a) strain EMCC073

A lyophilized vial of a sporulated culture of Bacillus thuringiensisEMCC0073 is inoculated into a 250 ml baffled shake flask containing 50ml nutrient broth in late afternoon and incubated for 12 hours at 30° C.with shaking at 250 rpm.

Volumes of 1.0 ml of the vegetative cells and 9.0 ml of 0.1M phosphatebuffer, pH 7 are injected into an "Isopac" vial containing 10 mg ofN-methyl-N'-nitro-N-nitroso-guanidine (NTG, Sigma Product # M6263). Themixture is incubated at 30° C. for 5, 10, and 20 minutes with low speedshaking. At each time point, a 0.5 ml sample is removed, diluted, andplated onto Nutrient Broth agar plates. The resulting plates areincubated at 30° C. for 3 days or longer before colonies are selectedfor microscopic examination. Semi-translucent colonies are selected andare grown in the medium described in Example 7. Two morphologicallydifferent Bacillus thuringiensis mutants, EMCC0123 and EMCC0124, areselected for bioassay as described infra in Example 8.

5.7. Example 7

Cultivation of Bacillus thuringiensis EMCC0073 mutants

Subcultures of Bacillus thuringiensis EMCC0073 and mutants thereof,EMCC0123 and EMCC0124, maintained on Nutrient Broth agar slants, areused to inoculate 250 ml baffled shake flasks containing 50 ml of mediumwith the following composition:

    ______________________________________    Corn Steep Liquor     15     g/l    Maltrin-100           40     g/l    Potato Starch         30     g/l    KH.sub.2 PO.sub.4     1.77   g/l    K.sub.2 HPO.sub.4     4.53   g/l    ______________________________________

The pH of the medium is adjusted to 7.0 with 10N NaOH.

After inoculation, the shake flasks are incubated at 30° C. on a rotaryshaker at 250 rpm for 72 hours until sporulation and cell lysisreleasing the crystals and spores are observed microscopically. Thewhole culture broths, obtained from the above fermentations, are used tocharacterize the insecticidal activity.

5.8. Example 8

Bioassay of crystal delta-endotoxins from Bacillus thuringiensis mutantsEMCCO123 and EMCCO124 against Spodoptera exigua and Heliothis zea

Standard artificial diet composed of water, agar, sugar, casein, wheatgerm, methyl paraben, sorbic acid, linseed oil, cellulose, salts, andvitamins is prepared by using methods known in the art. Whole brothsfrom the Bacillus thuringiensis mutants EMCC0123 and EMCC0124 asdescribed in Example 7 are diluted to 0.5 mg per ml and 1 mg per ml. A0.7 ml aliquot of molten diet is poured into each well of a plastic traybearing 40 individual wells and allowed to solidify. Aliquots of 50 μlof each diluted broth from the Bacillus thuringiensis mutants EMCC0123and EMCC0124 are added to the surface of the diet and allowed to dry.Three to six eggs each of Spodoptera exigua and Heliothis zea are thenapplied to the surface of the solidified diet. The trays are coveredwith a perforated sheet of clear mylar and placed on racks and incubatedfor 7 days at 28° C. and 65% humidity.

After 7 days, insect stunting and mortality are rated. Each tray isgiven a sharp blow against a table top, and larvae that did not move arecounted as dead. In the stunt scoring system, 4=full size larvae(control larvae), 3=3/4 size of control larvae, 2=1/2 size of controllarvae, 1=1/4 size of control larvae, and 0=no growth. The smaller thestunt score, the higher the activity of the Bacillus thuringiensis wholebroth preparation.

The results are shown in Table VI, infra. The insecticidal activities ofthe broths from the Bacillus thuringiensis mutants EMCC0123 and EMCC0124are significantly greater than that of the parent strain Bacillusthuringiensis EMCC0073 against both Heliothis zea and Spodoptera exigua.

                  TABLE VI    ______________________________________    Activity of Bacillus thuringiensis mutants    EMCC0123 and    EMCC0124 against Heliothis zea and Spodoptera exigua                            Heliothis zea              Spodoptera exigua       Stunt    Sample      % Mortality                          Stunt Score                                    % Mortality                                            score    ______________________________________    EMCC0123              1 mg  100       0       83      0.2            0.5 mg  100       0       50      0.5    EMCC0124              1 mg  100       0.5     100     0.5            0.5 mg  100       0.5     38      1.4    EMCC0073              1 mg   75       0.2     33      1.2            0.5 mg   50       0.5     17      1.8    ______________________________________

5.9. Example 9

Determination of size of CryIA(a) crystals from Bacillus thuringiensismutants EMCC0123 and EMCC0124

Crystal measurements are made by photographing spore/crystalpreparations with an Olympus BH2 microscope. Measurements of thecrystals in millimeters are made with a ruler, and then normalized tothe average length of the spores in each photo to account for anydifferences in photo enlargement. The volume for the bipyramidalcrystals is calculated using the following formula: V=(width²×length)/3. It is assumed that a mature endospore is approximately 1 μmin its longest diameter.

The results, shown in Table VII infra, indicate that the volumes of thecrystals of Bacillus thuringiensis mutant EMCC0123 and Bacillusthuringiensis mutant EMCC0124 are more than 2.2 times and 2.0 times,respectively, the volume of the crystal of the parent strain Bacillusthuringiensis EMCC0073.

                  TABLE VII    ______________________________________    Crystal dimensions of Bacillus thuringiensis    EMCC0073 mutants            Crystal         Crystal     Crystal            Length  Range   Width Range Volume                                              Number    Sample  (μm) (μm) (μm)                                  (μm)                                        (μm.sup.3)                                              Measured    ______________________________________    EMCC0123            1.85    1.7-2.0 1.55  1.4-1.7                                        1.48  5    EMCC0124            1.80    1.6-2.0 1.50  1.3-1.7                                        1.35  5    EMCC0073            1.65    1.5-1.8 1.10  1.0-1.2                                        0.67  5    ______________________________________

5.10. Example 10

Classical mutagenesis of Bacillus thuringiensis subsp. aizawai StrainEMCC0087

A lyophilized vial of a sporulated culture of Bacillus thuringiensissubsp. aizawai EMCC0087 is inoculated into 50 ml of Nutrient Brothsupplemented with 0.2% glucose in a 250 ml baffled shake flask andincubated overnight at 30° C. on a rotary shaker at 250 rpm. One ml ofthe overnight culture is inoculated into each of two 250 ml baffledshake flasks containing 50 ml of the same medium and incubated 3additional hours. The whole broths of the two shake flasks are thencombined. A 70 ml whole broth sample is centrifuged for 15 min at 12,000rpm (Sorvall SS-34 rotor), and the pellet is resuspended in 10.5 ml of0.1M phosphate buffer, pH 7.

A volume of 10 ml of the cell suspension in 0.1M phosphate buffer, pH 7is injected into an "Isopac" vial containing 10 mg ofN-methyl-N'-nitro-N-nitroso-guanidine (NTG, Sigma Product # M6263).After a 20 minute exposure time, a sample is removed, diluted, andplated on Nutrient Broth agar plates.

Plates are incubated at 30° C. for three days or longer before coloniesare selected for microscopic examination. One Bacillus thuringiensissubsp. aizawai mutant, EMCC0125, is selected based on crystal sizeobserved microscopically. The parent strain produces bipyramidalcrystals, while the mutant produces large bipyramidals and cylinders aswell as irregular shaped crystals.

Relative quantitation of toxin protein is done using SDS-PAGE, scanningthe gel and using integrated peak densities to determine the amount ofprotein relative to the parent strain Bacillus thuringiensis subsp.aizawai EMCC0087. The Bacillus thuringiensis subsp. aizawai mutantEMCC0125 produced 1.6 times more toxin per cell than the parent strainBacillus thuringiensis subsp. aizawai EMCC0087.

5.11. Example 11

Cultivation of Bacillus thuringiensis subsp. aizawai mutant EMCCO125

A subculture of Bacillus thuringiensis subsp. aizawai mutant EMCC0125,maintained on a Nutrient Broth agar plate, is used to inoculate a 250 mlbaffled shake flask containing 50 ml of the medium with the compositiondescribed in Example 7.

After inoculation, the shake flask is incubated at 30° C. on a rotaryshaker at 250 rpm for 88 hours. The whole culture broth is used tocharacterize the insecticidal activity.

5.12. Example 12

Bioassay of whole broth from Bacillus thuringiensis subsp. aizawaimutant EMCC0125 against Spodoptera exigua

The potency of the whole broth from Bacillus thuringiensis subsp.aizawai mutant EMCC0125 is determined by diet incorporation bioassayusing third instar Spodoptera exigua larvae.

The Bacillus thuringiensis subsp. aizawai mutant EMCC0124 whole brothfrom Example 11 is serially diluted to establish the range of potency.The parent strain, Bacillus thuringiensis subsp. aizawai EMCC0087cultivated as described in EXAMPLE 11, is also run.

Standard artificial diet composed of water, agar, sugar, casein, wheatgerm, methyl paraben, sorbic acid, linseed oil, cellulose, salts, andvitamins is prepared by using methods known in the art. The Bacillusthuringiensis subsp. aizawai mutant EMCC0125 whole broth is seriallydiluted to give 16 ml aliquots. Each aliquot is added to 184 g of moltendiet. The mixture is subsequently homogenized and then poured into aplastic tray bearing 40 individual wells. Once the diet had cooled andsolidified, one third instar Spodoptera exigua larva is added to eachwell, and the trays are covered with a perforated sheet of clear mylar.The trays are placed on racks and incubated for four days at 28° C. and65% humidity.

Per cent mortality is calculated after 5 days and the data is analyzedvia parallel probit analysis. LC50 values, LC90 values, the slope of theregression lines, and coefficient of variation (CV) are determined. LC50results are expressed in terms of colony forming units (cfu) tocompensate for variation in growth rates of the cultures. Samples arebioassayed a minimum of 3 times.

The results are shown in Table VIII, infra. The potency of the Bacillusthuringiensis subsp. aizawai mutant EMCCO125 is approximately 1.8 timesthat of the parent strain Bacillus thuringiensis subsp. aizawaiEMCC0087.

                  TABLE VIII    ______________________________________    Potency of Bacillus thuringiensis subsp.    aizawai mutant EMCC0125    on Spodoptera exigua    Sample     LC50     LC90        Slope                                         CV    ______________________________________    EMCC0087   1.7 × 10.sup.6                        6.3 × 10.sup.6                                    2.2  17.1    EMCC0125   9.5 × 10.sup.5                        3.5 × 10.sup.6                                    2.6  13.6    ______________________________________

6. DEPOSIT OF MICROORGANISMS

The following strains of Bacillus thuringiensis have been deposited inthe Agricultural Research Service Patent Culture Collection (NRRL),Northern Regional Research Center, 1815 University Street, Peoria, Ill.,61604, USA.

    ______________________________________    Strain    Accession Number                              Deposit Date    ______________________________________    EMCC0073  NRRL B-21014    November 16, 1992    EMCC0087  NRRL B-21147    October 6, 1993    EMCC0123  NRRL B-21387    January 18, 1995    EMCC0124  NRRL B-21388    January 18, 1995    EMCC0125  NRRL B-21389    January 18, 1995    ______________________________________

The following strains of Bacillus thuringiensis have been deposited withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroderweg 1b, D-3300 Braunschweig, Germany.

    ______________________________________    Strain    Accession Number                              Deposit Date    ______________________________________       NB 176-1              DSM 5480        August 10, 1989    NB 125    DSM 5526        September 14, 1989    ______________________________________

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122 and under conditions of the Budapest Treaty. The deposit representsa biologically pure culture of each deposited strain. The deposit isavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 1    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3826 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CCTGGGTCAAAAATTGATATTTAGTAAAATTAGTTGCACTTTGTGCATTTTTTCATAAGA60    TGAGTCATATGTTTTAAATTGTAGTAATGAAAAACAGTATTATATCATAATGAATTGGTA120    TCTTAATAAAAGAGATGGAGGTAACTTATGGATAACAATCCGAACATCAATGAATGCATT180    CCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGTGGAGAAAGAATAGAAACT240    GGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTT300    CCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGGGGAATTTTTGGTCCCTCT360    CAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAATAGAAGAATTC420    GCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCA480    GAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGATGCGT540    ATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTGGCAGTTCAA600    AATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCAGTT660    TTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCAATAGT720    CGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGATTATGCTGTGCGCTGGTAC780    AATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGATTGGGTAAGGTATAATCAA840    TTTAGAAGAGAGCTAACACTTACTGTATTAGATATCGTTGCTCTATTCTCAAATTATGAT900    AGTCGAAGGTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACGAACCCA960    GTATTAGAAAATTTTGATGGTAGTTTTCGTGGAATGGCTCAGAGAATAGAACAGAATATT1020    AGGCAACCACATCTTATGGATATCCTTAATAGTATAACCATTTATACTGATGTGCATAGA1080    GGCTTTAATTATTGGTCAGGGCATCAAATAACAGCTTCTCCTGTAGGGTTTTCAGGACCA1140    GAATTCGCATTCCCTTTATTTGGGAATGCGGGGAATGCAGCTCCACCCGTACTTGTCTCA1200    TTAACTGGTTTGGGGATTTTTAGAACATTATCTTCACCTTTATATAGAAGAATTATACTT1260    GGTTCAGGCCCAAATAATCAGGAACTGTTTGTCCTTGATGGAACGGAGTTTTCTTTTGCC1320    TCCCTAACGACCAACTTGCCTTCCACTATATATAGACAAAGGGGTACAGTCGATTCACTA1380    GATGTAATACCGCCACAGGATAATAGTGTACCACCTCGTGCGGGATTTAGCCATCGATTG1440    AGTCATGTTACAATGCTGAGCCAAGCAGCTGGAGCAGTTTACACCTTGAGAGCTCCAACG1500    TTTTCTTGGCAGCATCGCAGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACA1560    CAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCA1620    GGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAACCTTAAGA1680    GTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAGAATTCGCTACGCTTCTACT1740    ACAAATTTACAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGTAATTTTTCA1800    GCAACTATGAGTAGTGGGAGTAATTTACAGTCCGGAAGCTTTAGGACTGTAGGTTTTACT1860    ACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATGTCTTCAAT1920    TCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTTCCGGCAGAAGTAACCTTTGAG1980    GCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAAT2040    CAAATCGGGTTAAAAACAGATGTGACGGATTATCATATTGATCAAGTATCCAATTTAGTT2100    GAGTGTTTATCAGATGAATTTTGTCTGGATGAAAAACAAGAATTGTCCGAGAAAGTCAAA2160    CATGCGAAGCGACTTAGTGATGAGCGGAATTTACTTCAAGATCCAAACTTCAGAGGGATC2220    AATAGACAACTAGACCGTGGCTGGAGAGGAAGTACGGATATTACCATCCAAGGAGGCGAT2280    GACGTATTCAAAGAGAATTACGTTACGCTATTGGGTACCTTTGATGAGTGCTATCCAACG2340    TATTTATATCAAAAAATAGATGAGTCGAAATTAAAAGCCTATACCCGTTATCAATTAAGA2400    GGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGCTACAATGCAAAACAT2460    GAAACAGTAAATGTGCCAGGTACGGGTTCCTTATGGCCGCTTTCAGCCCAAAGTCCAATC2520    GGAAAGTGTGGAGAGCCGAATCGATGCGCGCCACACCTTGAATGGAATCCTGACTTAGAT2580    TGTTCGTGTAGGGATGGAGAAAAGTGTGCCCATCATTCGCATCATTTCTCCTTAGACATT2640    GATGTAGGATGTACAGACTTAAATGAGGACCTAGGTGTATGGGTGATCTTTAAGATTAAG2700    ACGCAAGATGGGCACGCAAGACTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTAGTA2760    GGAGAAGCGCTAGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAA2820    TTGGAATGGGAAACAAATATCGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTT2880    GTAAACTCTCAATATGATCAATTACAAGCGGATACGAATATTGCCATGATTCATGCGGCA2940    GATAAACGTGTTCATAGCATTCGAGAAGCTTATCTGCCTGAGCTGTCTGTGATTCCGGGT3000    GTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATAT3060    GATGCGAGAAATGTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTG3120    AAAGGGCATGTAGATGTAGAAGAACAAAACAACCAACGTTCGGTCCTTGTTGTTCCGGAA3180    TGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGTCGTGGCTATATCCTTCGT3240    GTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGCGTAACCATTCATGAGATCGAGAAC3300    AATACAGACGAACTGAAGTTTAGCAACTGCGTAGAAGAGGAAATCTATCCAAATAACACG3360    GTAACGTGTAATGATTATACTGTAAATCAAGAAGAATACGGAGGTGCGTACACTTCTCGT3420    AATCGAGGATATAACGAAGCTCCTTCCGTACCAGCTGATTATGCGTCAGTCTATGAAGAA3480    AAATCGTATACAGATGGACGAAGAGAGAATCCTTGTGAATTTAACAGAGGGTATAGGGAT3540    TACACGCCACTACCAGTTGGTTATGTGACAAAAGAATTAGAATACTTCCCAGAAACCGAT3600    AAGGTATGGATTGAGATTGGAGAAACGGAAGGAACATTTATCGTGGACAGCGTGGAATTA3660    CTCCTTATGGAGGAATAGTCTCATGCAAACTCAGGTTTAAATATCGTTTTCAAATCAATT3720    GTCCAAGAGCAGCATTACAAATAGATAAGTAATTTGTTGTAATGAAAAACGGACATCACC3780    TCCATTGAAACGGAGTGATGTCCGTTTTACTATGTTATTTTCTAGT3826    __________________________________________________________________________

What is claimed is:
 1. A method of obtaining a mutant of Bacillusthuringiensis having accession number NRRL B-21387, or NRRL B-21388,said mutant producing at least 1.25 times more volume of crystaldelta-endotoxin and having at least 1.25 times greater pesticidalactivity than a corresponding parental strain wherein the crystaldelta-endotoxin produced by the mutant Bacillus thuringiensis has anactivity directed towards the same pest as the crystal delta-endotoxinproduced by the corresponding parental strain, comprising(a) treatingthe parental strain with a mutagen; (b) culturing the mutagenized strainof step (a); and (c) selecting colonies from the cultured strain of step(b) with increased crystal delta-endotoxin production.
 2. The methodaccording to claim 1 in which the crystal volume of said mutant islarger than the crystal volume of said parental strain.
 3. The methodaccording to claim 1 in which the crystal volume of said mutant is atleast about 1.5 times the crystal volume of said parental strain.
 4. Themethod according to claim 1 in which the mutant produces a Cry Iprotein.
 5. The method according to claim 1 in which the mutant producesa CryIA(a)-like protein.
 6. The method according to claim 1 in which themutant is a mutant of Bacillus thuringiensis EMCC0073.
 7. The methodaccording to claim 1 in which the mutant is a mutant of Bacillusthuringiensis subsp. aizawai.
 8. The method according to claim 1 inwhich the parental strain is a wild-type strain.
 9. The mutant accordingto claim 1 in which the mutant shows a sporulation frequency at leasttwo logs lower than the sporulation frequency of the parental strain.10. The method according to claim 1 in which in step (i) the mutagen isselected from the group consisting of a chemical mutagen andelectromagnetic radiation.
 11. The method according to claim 1 in whichin step (i) the parental strain is treated with a chemical mutagenselected from the group consisting ofN-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate.
 12. Themethod according to claim 1 in which in step (i) the parental strain istreated with electromagnetic radiation selected from the groupconsisting of gamma-, X-ray, and UV-radiation.
 13. The method accordingto claim 1 in which the treated parental strain of (a) is cultured instep (b) in a medium suitable for the selection of asporogenous oroligosporogenous strains.
 14. The method according to claim 1 whichfurther comprises after step (b) and before step (c)(i) selectingtranslucent colonies from the cultured strain of (b); (ii) growing theselected translucent colonies of (c) in medium that does not fluidize onheating; (iii) deselecting asporogenous strains by subjecting thecolonies of (d) to a heat treatment; and (iv) growing the selectedcolonies remaining after step (e) in a culture medium.
 15. A method ofobtaining a mutant of Bacillus thuringiensis having accession numberNRRL B-21389, said mutant producing higher amounts of crystaldelta-endotoxin and having at least 1.25 times greater pesticidalactivity than a corresponding parental strain wherein the crystaldelta-endotoxin produced by the mutant Bacillus thuringiensis has anactivity directed towards the same pest as the crystal delta-endotoxinproduced by the corresponding parental strain, comprising(a) treatingthe parental strain with a mutagen; (b) culturing the mutagenized strainof step (a); and (c) selecting colonies from the cultured strain of step(b) with increased crystal delta-endotoxin production.